Kalindi Deshmukh

Differences in Collagen Metabolism between Normal and Osteoarthritic Human Articular Cartilage

Normal human articular cartilage synthesizes only one type of a chain, which exhibits the chromatographic behavior of the αl(II) chains described for chick and bovine cartilage. Osteoarthritic cartilage, on the other hand, synthesizes in addition a collagen containing α2 chains and β components. The different structural features of the two types of collagen may account for some of the functional defects of osteoarthritic cartilage.

Changes in collagen metabolism associated with the administration of penicillamine and various amino and thiol compounds

Abstract D-Penicillamine and BAPN (β-amino propionitrile) were found to be the most active agents able to cause an accumulation of neutral salt-soluble collagen in the skin of rats. Most analogues of penicillamine had very little or no effects. Blocking the α-amino group ( N -acetyl-penicillamine) or removal of the sulfhydryl group (valine) render inactive compounds. A higher homologue (mercaptoisoleucine) was ineffective. It would seem that the structure of D-penicillamine meets all the criteria for effectiveness, such as extracellular distribution, lack of metabolic deamination, sufficient amount of steric hindrance of the sulfhydryl group to retard its oxidation to disulfide, but yet not enough to prevent completely its reactivity. D-Penicillamine totally blocks the crosslinking of newly formed tropocollagen and is able to degrade a certain fraction of the more recently sythesized process insoluble collagen which has not yet completed its maturation D-Penicillamine affects equally both sexes, is more effective in young rats than in older ones and is not antagonized by copper if this metal is administered by a different route. If given simultaneously, mixed in the diet, the effect on collagen is inhibited and the toxicity of copper reduced, probably due to the marked affinity of both compounds and possible inactivation in the gastrointestinal tract. The urinary excretion of hydroxyproline in the D-penicillamine-treated rats falls within normal limits. Only the BAPN-treated animals showed an increase in urinary hydroxyproline. Since BAPN causes severe osseous abnormalities, whereas penicillamine does not, these findings would tend to support the view that an increase in urinary hydroxyproline is primarily a manifestation of abnormal metabolism of bone collagen.

Effects of lysosomal enzymes on the type of collagen synthesized by bovine articular cartilage

Abstract Bovine articular cartilage normally synthesizes a collagen containing three identical α-chains. After pre-incubation with rat liver lysosomal enzymes, it begins to synthesize significant amounts of the more ubiquitous collagen of the (α 1 ) 2 α 2 type. Since lysosomes are increased in osteoarthritis, it is possible that the abnormal biosynthetic patterns exhibited by cells in areas of degeneration are caused by such enzymes. Articular cartilage is an avascular tissue with very low cell density, composed primarily of extracellular substances such as collagen, proteoglycans, and glycoproteins. The structural integrity of this tissue depends on the relative proportion, nature, and structural organization of these components. Until recently, the destruction of cartilage seen in osteoarthritis was considered to result from a “wear and tear” process. This concept is not substantiated by recent ultrastructural and biochemical findings. Cellular activity in the involved areas leads to enlarged clones of chondrocytes containing increased numbers of intracellular organelles reflecting synthetic and secretory activity (1). There is an inverse correlation between the severity of the degenerative changes and the glycosaminoglycan content of the tissue (2–7). On the other hand, radioactive sulfate incorporation increases in osteoarthritis, an indication of the attempts made by the cells involved to repair the lesion (2). The nature of the proteoglycans synthesized under these conditions (less keratan sulfate and more chondroitin-4-sulfate) reflect the behaviour of immature chondroblasts (3–8). Lysosomal proteases have been associated with the degradation of the matrix (9–12). Cathepsin-D and a neutral protease which degrade proteoglycans at pH 5.0 and 7.0 respectively are considerably increased in early osteoarthritic lesions (13–15). Although the collagen content of cartilage does not change in osteoarthritis, qualitative differences may exist. Recently, we have shown that whereas normal human cartilage synthesizes only cartilage type collagen or (α 1 -Type II) 3 , osteoarthritic cartilage synthesizes in addition significant amounts of (α 1 ) 2 α 2 collagen (skin type) (16). Articular cartilage collagen is quite different from other ubiquitous forms of mammalian collagens. In addition to containing three identical α-chains, it has four to five times more hydroxylysine and glycosidically associated carbohydrate than collagen from other tissues (17). It is quite possible that the abnormal collagen deposited by the cells at the site of degeneration may give rise to a mechanically weaker structure and lead to a loss of cartilage. While attempting to elucidate the mechanism underlying this abnormal metabolic pattern, it became apparent that lysosomal enzymes can alter the function of normal cartilage cells causing them to synthesize non-specific collagen molecules.

Turnover and age distribution of a collagen fraction extractable from rat skin by mercaptoethylamine

Abstract Thiol compounds such as 0.2 M cysteamine at neutral pH and at 4 ° are able to solubilize significant amounts of dermal insoluble collagen. In young rats (2 months old or younger) 90% of the insoluble collagen can be extracted by 0.2 M cysteamine. In older animals the proportion of insoluble residue increases. Two successive extractions of insoluble collagen with 0.2 M cysteamine yielded 33% β and 50% β components, respectively, an indication that the material which is more resistant to solubilization may be more crosslinked. This increased crosslinking seems to be related to the biological age of the material, a conclusion substantiated by the slower turnover rate of the less readily soluble fraction. The native collagen solubilized by cysteamine shows the same sedimentation properties and electrophoretic mobility as salt soluble collagen, but yields different proportions of α and β components Following thermal denaturation.

Effects of calcitonin and parathyroid hormone on the metabolism of chondrocytes in culture

Rabbit articular chondrocytes in suspension culture synthesize Type II collagen [3alpha1(II)] in the absence of extracellular Ca2+ and Type I collagen [2alpha1(I) - alpha2] in the complete medium. As a result of pre-treatment in monolayer culture with calcitonin or parathyroid hormone in the complete medium, an influx of Ca2+ into the cells occurs. These cells produce mainly Type I collagen when transferred to suspension cultures in the medium devoid of CaCl2. If added directly to the suspension culture medium containing no CaCl2, calcitonin stimulates an active efflux of Ca2+ from the cells into the medium and leads the cells to synthesize Type I collagen. Under similar conditions, parathyroid hormone does not change the collagen-phenotype.

Identification of stable intermolecular crosslinks present in reconstituted native collagen fibers.

Abstract Soluble collagen extracted by 0.2 M cysteamine from rat skin contains 2.5 to 2.7 residues of peptide bound α-aminoadipic semi-aldehyde per α-chain. When reconstituted into fibers and maintained at 37° C these aldehydes become involved in the formation of stable non-reducible crosslinks. Using collagen biosynthetically labeled at the lysine and hydroxylysine positions, these stable crosslinks have been partially identified. Lysinonorleucine and its hydroxyderivative account for 50% of the initial aldehyde residues present on the collagen molecule while two unidentified peaks (post-lysine and post-histidine) account for the other 50%.

A Comparative Study of the Crosslinking Precursors Present in Rat Skin and Tail Tendon Collagen

Tissue specific differences in the amounts and the location of aldehyde groups have been observed which are directly related to the solubility characteristics of collagen, extent of maturation and the functional role of the tissue. Whereas the aldehydes are restricted to the N-terminal region of rat tail tendon collagen, in skin they are also present in the helical region of the molecule and are able to participate in the formation ofstableinter-molecular crosslinks.

In vitro formation of crosslinks in mature rat skin collagen in the absence of N-terminal peptides

Abstract Soluble collagen extracted by cysteamine from rat skin contains 1.7 to 2.0 moles of peptide bound α-amino adipic semi-aldehyde along the helical portion of the α-chains. Removal of the N-terminal peptides by limited CNBr cleavage yields a collagen that has native characteristics (viscosity and optical rotation) and forms regular 640 A banded fibers. Incubation of this material at 37° C. gives rise to Schiff base type crosslinks involving these aldehydes and lysine or OH-lysine of neighboring molecules.

Synthesis of Tissue Nonspecific Collagen by Bovine Articular Cartilage as a Result of Aging in Vitro

Summary Bovine articular cartilage synthesizes collagen containing three identical chains, α l(II). Treatment with rat liver lysosomes or certain individual enzymes changes this synthetic pattern to a fibroblast-type (αl)2.α2 collagen. Similar alteration can also be observed after prolonged incubation of cartilage in vitro without addition of any exogenous factors or in the presence of vitamin A, which is known to be a lysosomal labilizer. It is possible that the endogenous lysosomal activity is released by aging in vitro, leading to the dedifferentiation of chon-drocytes. This research was supported by NIH Special Fellowship No. AM-53414. The author greatfully acknowledges the helpful criticism by Dr. M. E. Nimni and thanks Dr. Marvin Meyers for providing the surgical specimens.